Inflammatory bowel disease (IBD) is characterized by relapsing intestinal inflammation and altered epithelial permeability resulting in fluid/electrolyte loss and systemic exposure to luminal antigens. Permeability changes have been attributed to defective tight junction (TJ) function. The TJ is intimately affiliated with a subjacent adherens junction and they have been collectively referred to as the Apical Junctional Complex (AJC). The AJC, in turn, is in close proximity to underlying desmosomes. Functional orchestration of these intercellular junctions is required for appropriate cell-cell adhesion and regulation of paracellular permeability. The major objectives of this proposal are to identify relevant structural elements in the AJC, define their associations and analyze their intracellular trafficking. Specific Aim 1 will identify and characterize membrane raft associated intercellular junction proteins important in regulating cell-cell adhesion and paracellular permeability. AJC enriched membrane rafts were used to generate monoclonal antibodies that recognize unique epitopes in intercellular junctions of intestinal epithelial cells. Three candidate monoclonal antibodies were identified. We established identitity of one of these antigens as a novel truncated variant of a cadherin, referred to as desmoglein 2 (Dsg2). We will elucidate the role of Dsg2/tDs2 in regulating intestinal epithelial intercellular association and AJC function. Identity of the other two antigens will be established in future studies. Specific Aim 2 will define intracellular trafficking pathways utilized by AJC proteins. We will further explore our initial studies suggesting that AJC disassembly &barrier disruption induced by exposure of epithelial cells to the proinflammatory cytokine, IFNgamma is associated with select endocytosis of key AJC proteins by macropinocytosis. IFNgamma induced intracellular trafficking of AJC proteins will be further explored. Specific Aim 3 will explore the role of extracellular domains of claudin(s) in regulating paracellular permeability. Studies will focus on using biotinylated and photoactive bait peptides emulating distinct extracellular domains of claudin(s). Peptide- Protein complexes will be isolated and analyzed. Functional consequences of peptide association with claudin(s) extracellular domains on AJC structure and paracellular permeability will be examined. Information from these studies will provide important mechanistic insight into AJC structure/function and should provide novel insights into potential therapeutic targets for correction of increased permeability in IBD. Additionally, understanding the molecular composition of AJCs will also facilitate in designing molecules that can be delivered via the paracellular route.